The present invention relates to the discovery that certain tripeptide amides, which correspond to viral capsid sequences, can be used to inhibit viral infection, including human immunodeficiency virus (HIV) infection. More specifically, medicaments comprising these tripeptide amides and methods of using said compounds for the prevention and treatment of viral infection, such as HIV infection, are provided.
All viruses are composed of a protein shell surrounding a nucleic acid containing core. The protein shell directly surrounding the viral nucleic acid is called a capsid, whereas, the complete protein-nucleic acid complex having both the capsid and the nucleic acid is called a nucleocapsid. Arenaviruses, rotaviruses, orbiviruses, retroviruses (including lentiviruses), papillomaviruses, adenoviruses, herpesviruses, paramyxovirus, myxovirus, and hepadnaviruses all exhibit these general structural features. (Virology, Fields ed., third edition, Lippencott-Raven publishers, pp 1513, 1645,1778, 2047, 2113, 2221, and 2717 (1996)).
The capsid is composed of many subunits (capsomeres) and capsomeres are formed from several homo- or hetero-polymers of protein. The noncovalent bonds between capsomeres in a viral assembly are of the same sort that stabilize a folded protein domain. The interface between two subunits can look very much like a single domain, with amino acid side chains tightly packed against one another. A common feature to most of the virus structures analyzed is the way in which a polypeptide chain from one capsomere can extend under or over domains of neighboring capsomeres. These extended polypeptide arms intertwine with other polypeptide arms and help to stabilize the capsid by initiating hydrophobic interactions, hydrogen bonding, and salt bridges. Contacts between individual capsomeres, and for some viruses also contacts with core proteins, determine the overall capsid structure and if a number of identical capsomeres are involved, repeated contacts occur and the resulting structure is symmnetrical. (Id. at 62).
Some simple viruses form spontaneously from their dissociated components while others require enzyme-catalyzed modifications of the capsomeres to trigger assembly. Viral self assembly is driven by the stability of the interactions between protein subunits under conditions that favor association. More complex viruses are often constructed from subassemblies that have undergone self assembly processes. (Id. at pp 62, 70, 1646 and 1888). Although the capsids of many viruses differ in protein composition, a general viral structural design has evolved characterized by polymerized capsomeres that, in turn, are composed of several homo- or hetero-polymers of protein.
HIV is the name given to a lentivirus that infects humans and that causes acquired immuno-deficiency syndrome (AIDS). The lentivirus isolates from humans are grouped into one of two types (HIV-1 and HIV-2) on the basis of serologic properties and sequence analysis of molecularly cloned viral genomes. Genetically distinct lentiviruses have been obtained from several non-human primate species including African green monkeys, sooty magabeys, mandrills, chimpanzees, and sykes. Collectively, the lentivirus isolates from non-human primates are called SIV. Sequence analysis reveals that the genomes of some SIV strains and HIV-1 and HIV-2 strains exhibit a high degree of homology. Further, electron microscopy reveals that the ultrastructure of HIV and SIV are similar in that both have virions about 110 nm in diameter with a cone-shaped nucleocapsid surrounded by a lipid bilayer membrane that contains envelope glycoprotein spikes. (Id. at pp.1882-1883).
HIV is a complex retrovirus containing at least seven genes. The viral structural genes, designated gag, pol, and env, respectively code for the viral core proteins, reverse transcriptase, and the viral glycoproteins of the viral envelope. The remaining HIV genes are accessory genes involved in viral replication. The gag and env genes encode polyproteins, i.e., the proteins synthesized from each of these genes are post-translationally cleaved into several smaller proteins.
Although the overall shape of HIV and SIV virions is spherical, the nucleocapsid is asymmetrical having a long dimension of about 100 nm, a wide free end about 40-60 nm, and a narrow end about 20 nm in width. The nucleocapsid within each mature virion is composed of two molecules of the viral single-stranded RNA genome encapsulated by proteins proteolytically processed from the Gag precursor polypeptide. Cleavage of the gag gene polyprotein Pr55gag by a viral coded protease (PR) produces mature capsid proteins. These gag gene products are the matrix protein (p17), that is thought to be located between the nucleocapsid and the virion envelope; the major capsid protein (p24), that forms the capsid shell; and the nucleocapsid protein (p9), that binds to the viral RNA genome. This proteolytic processing in infected cells is linked to virion morphogenesis. (Id. at pp 1886-1887).
The major capsid protein p24 (also called CA) contains about 240 amino acids and exhibits a molecular weight of 24-27 kD. The protein p24 self-associates to form dimers and oligomeric complexes as large as dodecamers. Genetic studies with mutations in the HIV-1 gag polyprotein have identified several functional domains in the p24 protein including the C terminal half of the molecule and a major homology region (MHR) spanning 20 amino acids that is conserved in the p24 proteins of diverse retroviruses. These mutations appear to affect precursor nucleocapsid assembly. (Id. at pp 1888-1889).
Since the discovery of HIV-1 as the etiologic agent of AIDS, significant progress has been made in understanding the mechanisms by which the virus causes disease. While many diagnostic tests have been developed, progress in HIV vaccine therapy has been slow largely due to the heterogeneous nature of the virus and the lack of suitable animal models. (See, e.g., Martin, Nature, 345:572-573 (1990)).
A variety of pharmaceutical agents have been used in attempts to treat AIDS. Many, if not all, of these drugs, however, create serious side effects that greatly limit their usefulness as therapeutic agents. HIV reverse transcriptase is one drug target because of its crucial role in viral replication. Several nucleoside derivatives have been found to inhibit HIV reverse transcriptase including azidothymidine (AZT, zidovidine(copyright)). AZT causes serious side effects such that many patients cannot tolerate its administration. Other nucleoside analogs that inhibit HIV reverse transcriptase have been found to cause greater side effects than AZT. Another drug target is the HIV protease (PR) crucial to virus development. PR is an aspartic protease and can be inhibited by synthetic compounds. (Richards, FEBS Lett., 253:214-216 (1989)). Protease inhibitors inhibit the growth of HIV more effectively than reverse transcriptase inhibitors but prolonged therapy has been associated with metabolic diseases such as lipodystrophy, hyperlipidemia, and insulin resistance.
Additionally, HIV quickly develops resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors and protease inhibitors. This resistance can also spread between patients. Studies have shown, for example, that one tenth of the individuals recently infected by HIV already have developed resistance to AZT, probably because they were infected by a person that at the time of transmission carried a virus that was resistant to AZT.
It would be useful in the treatment and prevention of viral infections, including HIV and SIV, to have specific and selective therapeutic agents that cause few, if any, side effects.
The present invention is related to tripeptide amides that inhibit viral infectivity. An intact capsid structure is of vital importance for the infectivity of a virion. A way to disrupt assembly of capsid protein macromolecules, that for their infectivity are dependent on di-, tri-, tetra-, or poly-merization, is to construct small molecules that affect such protein-protein interactions. It was discovered that tripeptides with their carboxyl terminus hydroxyl group replaced with an amide group have such an inhibiting effect on capsid-protein interactions. Thus, aspects of the present invention relate to tripeptide amides that affect viral capsid assembly.
In desirable embodiments, the tripeptide amides bind to a protein that is involved in capsomere organization and capsid assembly of HIV-1, HIV-2, and SIV and thereby inhibit and/or prevent proper capsid assembly and, thus, viral infection. The tripeptide amides Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2 are the preferred species. These tripeptide amides and peptidomimetics resembling their structure (collectively referred to as xe2x80x9cpeptide agentsxe2x80x9d) are used in a monomeric or multimeric form. The tripeptide amides are suitable for therapeutic and prophylactic application in mammals, including man, suffering from viral infection.
In one embodiment, a composition for inhibiting viral replication in host cells infected with a virus has an effective amount of a peptide in amide form selected from the group of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, GIn-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. In some embodiments, the compositions described above are joined to a support and in other embodiments, the compositions described above are incorporated into a pharmaceutical having a pharmaceutically acceptable carrier. For example, the peptide in amide form can have the formula Ser Ile-Leu-NH2 and can be joined to a support.
Methods of inhibiting viral replication in a host cell are also embodiments of the present invention. One approach, for example, involves administering to a cell an effective amount of a peptide in amide form selected from the group consisting of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. The method described above can be supplemented with an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. The tripeptide amide used in the method above can be joined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another embodiment, a composition for inhibiting HIV replication in host cells includes an effective amount of a peptide in amide form selected from the group consisting of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. In some embodiments, these tripeptide amides are joined to a support and in other embodiments, these peptides are incorporated into a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another method, an approach to inhibit HIV replication in host cells is provided, which involves administering to said cells an effective amount of a peptide in amide form selected from the group consisting of peptides of the formula Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. This method can also be supplemented by an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. Further, the tripeptide amide used in this method can be joined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.
In another method, an approach for interrupting viral capsid assembly is provided. This approach involves contacting a cell with an effective amount of a peptide in amide form selected from the group consisting of peptides of the formula Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. The tripeptide amide can be joined to a support or incorporated in a pharmaceutical.
In still another method, an approach for interrupting HIV capsid assembly is provided. This approach also involves contacting a cell with an effective amount of a peptide in amide form selected from the group consisting of peptides of the formula Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. The tripeptide amide of this method can be joined to a support or incorporated in a pharmaceutical.
Methods of identification of peptide agents that inhibit viral replication, specifically HIV replication are also provided. By one method, for example, a peptide agent for incorporation into an anti-viral pharmaceutical is identified by contacting a plurality of cells infected with a virus with an effective amount of a peptide agent, analyzing the virus for incomplete capsid formation, and selecting the peptide agent that induces incomplete capsid formation. This method can involve an analysis of capsid formation that employs microscopy and the virus can be selected from the group consisting of HIV-1, HIV-2, and SIV. Further, the peptide agent identified can be selected from the group consisting of a tripeptide amide and a peptidomimetic resembling a tripeptide amide. For example, the peptide agent above can be selected from the group consisting of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. In a preferred embodiment, the peptide agent used in the method above has an amino acid sequence that corresponds to an amino acid sequence of p24.
In another embodiment, a method of identifying a peptide agent that binds to a viral protein is provided. Some aspects of this method involve providing a viral protein, contacting the viral protein with an effective amount of a peptide agent, and detecting the formation of a complex comprising the viral protein and the peptide agent. Some methods use a viral protein that is from a virus selected from the group consisting of HIV-1, HIV-2, and SIV. Further, in some embodiments, the peptide agent is selected from the group consisting of a tripeptide amide and a peptidomimetic resembling a tripeptide amide. Desirably, the method above employs a peptide agent selected from the group consisting of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. Additionally, a method of making a pharmaceutical is provided in which the peptide agent identified by the methods above are incorporated in a pharmaceutical.
Another approach to making a pharmaceutical involves administering to a cell an effective amount of a peptide in amide form, described above, detecting an inhibition of viral replication in the cell, and incorporating the peptide that causes inhibition of viral replication into the pharmaceutical. This method can involve the use of a tripeptide amide selected from the group consisting of Ser-Ile-Leu-NH2, Ile-Leu-Asp-NH2, Gly-Pro-Lys-NH2, Pro-Lys-Glu-NH2, Lys-Glu-Pro-NH2, Glu-Pro-Phe-NH2, Arg-Asp-Tyr-NH2, Asp-Tyr-Val-NH2, Tyr-Lys-Thr-NH2, Arg-Ala-Glu-NH2, Ala-Glu-Gln-NH2, Glu-Gln-Ala-NH2, Val-Lys-Asn-NH2, Thr-Glu-Thr-NH2, Leu-Leu-Val-NH2, Val-Gln-Asn-NH2, Gln-Asn-Ala,-NH2, Asn-Ala-Asn-NH2, Asn-Pro-Asp-NH2, Pro-Asp-Cys-NH2, Cys-Lys-Thr-NH2, Thr-Ile-Leu-NH2, Pro-Gly-Ala-NH2, Thr-Leu-Glu-NH2, Thr-Ala-Cys-NH2, Ala-Cys-Gln-NH2, Gln-Gly-Val-NH2, Pro-Gly-His-NH2, and Arg-Val-Leu-NH2. Further, this method can be supplemented with administration of an antiviral compound selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors into the pharmaceutical. Additionally, the method above can be supplemented by incorporating a carrier into the pharmaceutical.
In another embodiment, a composition for inhibiting viral replication in host cells infected with a virus includes an effective amount of a peptide of the formula X1X2X3xe2x80x94R, wherein X1, X2, and X3 are any amino acid, wherein R is a modulation group attached to the carboxy-terminus of said peptide and R comprises an amide group or other moiety having similar charge and steric bulk and wherein said composition inhibits viral replication by interrupting viral capsid assembly. This composition can be a peptide selected from the group consisting of peptides having the formula Ser-Ile-Leu-R, Ile-Leu-Asp-R, Gly-Pro-Lys-R, Pro-Lys-Glu-R, Lys-Glu-Pro-R, Glu-Pro-Phe-R, Arg-Asp-Tyr-R, Asp-Tyr-Val-R, Tyr-Lys-Thr-R, Arg-Ala-Glu-R, Ala-Glu-Gln-R, Glu-Gln-Ala-R, Val-Lys-Asn-R, Thr-Glu-Thr-R, Leu-Leu-Val-R, Val-Gln-Asn-R, Gln-Asn-Ala,-R, Asn-Ala-Asn-R, Asn-Pro-Asp-R, Pro-Asp-Cys-R, Cys-Lys-Thr-R, Thr-Ile-Leu-R, Ala-Leu-Gly-R, Pro-Gly-Ala-R, Thr-Leu-Glu-R, Thr-Ala-Cys-R, Ala-Cys-Gln-R, Gln-Gly-Val-R, Pro-Gly-His-R, and Arg-Val-Leu-R. Desirably, X3 is glycine in these embodiments.